BJ-PI2, A non-hemorrhagic metalloproteinase from Bothrops jararaca snake venom

Biochimica et Biophysica Acta 1820 (2012) 1809–1821

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BJ-PI2, A non-hemorrhagic metalloproteinase from Bothrops jararaca snake venom Igor Rapp Ferreira da Silva a, Raquel Lorenzetti a, André Lisboa Rennó a, Lineu Baldissera Jr. a, André Zelanis b, Solange Maria de Toledo Serrano b, Stephen Hyslop a,⁎ a Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, Cidade Universitária Zeferino Vaz, Campinas, 13083‐887, SP, Brazil b Laboratório Especial de Toxinologia Aplicada-CAT/CEPID, Instituto Butantan, Avenida Brazil 1500, 05503‐900 São Paulo, SP, Brazil

a r t i c l e

i n f o

Article history: Received 5 January 2012 Received in revised form 7 July 2012 Accepted 25 July 2012 Available online 31 July 2012 Keywords: Bothrops jararaca Coagulopathy Hemorrhage Myonecrosis Snake venom metalloproteinase (SVMP) Vascular permeability

a b s t r a c t Background: Envenoming by Bothrops jararaca can result in local pain, edema, hemorrhage and necrosis, partially mediated by snake venom metalloproteinases (SVMPs). Here, we describe the characterization of BJ-PI2, a P-I class SVMP from B. jararaca venom, and its local tissue actions. Methods: BJ-PI2 was purified by a combination of gel filtration, anion-exchange chromatography and reverse phase HPLC, and identified by mass spectrometry. Clotting and fibrin(ogen)olytic activities were assayed using conventional methods. Hemorrhagic activity and changes in vascular permeability were examined in rat dorsal skin. Myonecrosis and inflammatory activity were examined in mouse gastrocnemius muscle. Results: BJ-PI2 was a 23.08 kDa single-chain polypeptide. Tryptic fragments showed highest homology with SVMP insularinase A from Bothrops insularis, but also with B. jararaca SVMP bothrojaractivase; less similarity was observed with B. jararaca SVMPs BJ-PI and jararafibrases II and IV. BJ-PI2 did not clot fibrinogen or rat citrated plasma but had α- and β-fibrinogenolytic activity (inhibited by EDTA and 1,10-phenanthroline but not by PMSF) and attenuated coagulation after plasma recalcification. BJ-PI2 had fibrinolytic activity. BJ-PI2 increased the vascular permeability of rat dorsal skin (inhibited by 1,10-phenanthroline). BJ-PI2 was not hemorrhagic or myonecrotic but caused migration of inflammatory cells. In contrast, venom was strongly hemorrhagic and myonecrotic but caused less infiltration of inflammatory cells. Conclusions: BJ-PI2 is a non-hemorrhagic, non-myonecrotic, non-coagulant P-I class SVMP that may enhance vascular permeability and inflammatory cell migration in vivo. General significance: BJ-PI2 contributes to enhanced vascular permeability and inflammatory cell migration after envenoming, but not to venom-induced hemorrhage and necrosis. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Snake venom metalloproteinases (SVMPs) belong to the M12 family of proteases and are subdivided into three classes: P-I, P-II and P-III [1]. P-I SVMPs consist of proteins containing only a metalloproteinase domain while P-II and P-III SVMPs contain metalloproteinase and disintegrin domains, and metalloproteinase, disintegrin, cysteine-rich and C-type lectin domains, respectively. The metalloproteinase domain consists of a conserved amino acid sequence HEXXHGXXH that may provide the hydrophobicity needed to immobilize a zinc atom [2]. SVMPs are associated with hemostatic disturbances, primarily blood incoagulability through interference with the coagulation cascade, and local and systemic hemorrhage through damage to blood vessels and thrombosis [2–4]. Transcriptomic and proteomic studies have shown that metalloproteinases are the most abundant components in Bothrops venom glands and venoms (see [5] and references therein), which ⁎ Corresponding author. Tel.: +55 19 3521 9536; fax: +55 19 3289 2968. E-mail address: [email protected] (S. Hyslop). 0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2012.07.011

suggest an important role for these SVMPs in envenoming by these snakes. Indeed, part of the local pain, edema, inflammation, hemorrhage and necrosis, as well as systemic effects such as coagulopathy and systemic hemorrhage seen after envenoming by Bothrops snakes [6–9] have been associated with the action of SVMPs [10–14]. SVMPs, such as BaP1, a metalloproteinase from Bothrops asper venom, exert many of their local effects by activating inflammatory immune cells to release pro-inflammatory cytokines (IL-1β and IL-6) [15] and mediators such as prostaglandins, e.g., PGE2 [16,17]. Several metalloproteinases have been isolated from Bothrops jararaca venom, the most studied so far being jararhagin [18], a 52 kDa P-III SVMP with fibrino(geno)lytic and hemorrhagic activity initially purified and characterized by Maruyama et al. [19]. Jararhagin degrades extracellular matrix (ECM) proteins such as laminin, fibronectin and type IV collagen, stimulates the production of IL-1β, IL-6 and TNF-α and inhibits collagendependent platelet aggregation and von Willebrand factor (vWF) degradation [18]. Other P-III SVMPs isolated from B. jararaca venom include hemorrhagic factor 3 (HF3) [20–26] and bothropasin [24,27–29]. Some P-I SVMPs have also been isolated from B. jararaca venom. Maruyama et al. [19,30] studied a group of P-I SVMPs collectively known as

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A

B

1

2

1

kDa

3

4

97 66 45 30

2

20.1

14.4

C

D

1

kDa

2

97 66 45 30

20.1 14.4

E

F kDa

97 66 45 30

20.1 14.4

M

1

2

3

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jararafibrases (~20–21 kDa) with fibrin(ogen)olytic activity and weak hemorrhagic activity that could be inhibited by EDTA and 1,10phenanthroline. These SVMPs also degraded ECM proteins such as laminin, fibronectin and type IV collagen. More recently, two other P-I SVMPs have been purified from this venom: BJ-PI, a highly proteolytic (caseinolytic) enzyme with no hemorrhagic activity in mice [24], and bothrojaractivase [31], which has strong fibrino(geno)lytic activity and is a prothrombin activator. Although the enzymatic activity of these enzymes is relatively well studied their actions in vivo remain unclear. In this work, we describe the purification and characterization from B. jararaca venom of BJ-PI2, a non-hemorrhagic, non-myonecrotic metalloproteinase with fibrin(ogen)lytic activity that increases vascular permeability and stimulates the migration of inflammatory cells into damaged tissue. This protein may contribute to the local edema and inflammatory response seen after envenoming by B. jararaca. 2. Materials and methods 2.1. Reagents and venom Bovine serum albumin, casein, elastin-Congo red, fibrinogen (bovine, plasminogen-free), n-t-boc-L-alanine p-nitrophenyl ester, 1,10-phenanthroline and human thrombin were from Sigma Chemical Co. (St. Louis, MO, USA). Phenylmethylsulphonyl fluoride (PMSF) was from Calbiochem (La Jolla, CA, USA) and EDTA was from Mallinckrodt-Baker (Phillipsburg, NJ, USA). Low-melting point agarose was from USB (Santa Clara, CA, USA). Reagents for electrophoresis, molecular mass markers and column chromatography media (Superdex 75 and Sepharose Q) were from GE Healthcare Life Sciences (Piscataway, NJ, USA). Isoflurane and sodium thiopental were from Cristália (Itapira, SP, Brazil). Other reagents were of analytical grade obtained from local suppliers. Venom from adult B. jararaca snakes of both sexes was obtained from the Centro de Extração de Toxinas Animais (CETA, Morungaba, SP, Brazil) and stored lyophilized at − 20 °C. 2.2. Animals Male Wistar rats (300–400 g) and male Swiss mice (20–30 g) were obtained from the Multidisciplinary Center for Biological Investigation (CEMIB) at UNICAMP. The animals were housed in standard plastic cages with a wood shaving substrate (5/cage for rats and 10/cage for mice) at 23 °C on a 12 h light/dark cycle (lights on at 6 a.m.) and free access to food and water. The animal protocols were approved by an institutional Committee for Ethics in Animal Use (CEUA/UNICAMP, protocol no. 2253–1) and the experiments were done according to the general ethical guidelines for animal use established by the Brazilian Society of Laboratory Animal Science (SBCAL) and EC Directive 86/609/EEC for Animal Experiments. 2.3. Purification protocol Venom (100 mg) dissolved in 2 ml of 0.01 M Tris–HCl, pH 8.0, containing 0.01 M CaCl2 and 0.15 M NaCl was centrifuged (12,000 g, 12 min, 4 °C) and the supernatant was applied to a column

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(16 cm× 60 cm) of Superdex 75 previously equilibrated with the same buffer at 18 °C. The column was eluted at a flow rate of 0.5 ml/min and the elution profile was monitored at 280 nm using an ÄKTAprime chromatographic system. The fractions were assayed for caseinolytic activity and those corresponding to the second major peak containing caseinolytic activity were pooled and concentrated by centrifugation (3000 g, 30 min, 4 °C) in Amicon ultrafilters (Millipore) to remove NaCl prior to the next step. The enzyme concentrate obtained in the previous step was diluted with 0.01 M Tris–HCl, pH 8.0, containing 0.01 M CaCl2 and applied to a 5 ml HiTrap Sepharose Q column equilibrated with the same buffer. The column was washed (0.5 ml/min) with Tris–HCl/Ca 2+ buffer to remove unbound proteins and bound proteins were then eluted with a linear gradient (0–0.5 M) of NaCl and the elution profile was monitored at 280 nm. Fractions corresponding to the peak with caseinolytic activity that eluted before the gradient were pooled and concentrated by centrifugation (3000 g, 30 min, 4 °C) in Amicon ultrafilters (Millipore) to remove NaCl prior to the next step. Reverse-phase HPLC (RP-HPLC) of the peak from the previous step was done using a Jupiter C18 column (250 mm × 4.6 mm × 5 μm; 300 Å; Phenomenex, Torrance, CA, USA) coupled to a Shimadzu chromatographic system that consisted of two pumps (LC10AD VP), a UV/Vis detector (SPD-10A), a fraction collector (FRC-10A) and a system controller (SCL-10A VP). The column was initially equilibrated with 0.1% trifluoroacetic acid (TFA). The sample was dissolved in H2O with 0.1% TFA. Proteins were eluted with a linear gradient (0–100%) of 99.9% acetonitrile in 0.1% TFA and the elution profile was monitored at 280 nm. The purity of the protein was assessed by SDS-PAGE and mass spectrometry. 2.4. Sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was done in a discontinuous system with a 4% polyacrylamide stacking gel prepared in 0.5 M Tris–HCl, pH 6.8, containing 0.4% SDS, and a 15% running gel prepared in 1.5 M Tris, pH 8.8, containing 0.4% SDS [32]. The samples were diluted in sample buffer containing 4% bromophenol blue, 0.06 M Tris–HCl, 2% SDS and 10% glycerol and boiled for 5 min prior to loading onto the stacking gel. In some cases, 0.1% dithiothreitol or β-mercaptoethanol was added to the samples before boiling. The gels (10 cm × 12.5 cm) were run in a mini-VE 206E system (GE LifeSciences) at constant voltage (100 V), with 0.625 M Tris–HCl, 1.92 M glycine and 1% SDS, pH 6.8, as the running buffer. Molecular mass markers were included in all runs. After electrophoresis, the gels were stained either with Coomassie brilliant blue or silver nitrate and documented. 2.5. 2D-polyacrylamide gel electrophoresis Venom (300 μg) or BJPI-2 (50 μg) was dissolved in 250 μl of rehydration solution (7 M urea, 2 M thiourea, 2% CHAPS, 0.5% immobilized pH gradient (IPG) buffer (GE Healthcare Life Sciences) and 1% bromophenol blue) and precast IPG isoelectric focusing (IEF) strips (13 cm, pH 3–10 linear) were soaked in this solution prior to the first dimension of electrophoresis (IEF) in a Multiphor II System (GE Healthcare Life Sciences),

Fig. 1. Purification of BJ-PI2 from B. jararaca venom. (A) Gel filtration (Superdex 75) elution profile of B. jararaca venom showing the two major peaks (1 and 2) with caseinolytic activity. The column was equilibrated and eluted (0.5 ml/min) with 0.01 mM Tris–HCl, pH 8.0, containing 0.01 M CaCl2 and 0.15 M NaCl. The elution profile was monitored at 280 nm. (■) protein, (●) caseinolytic activity. (B) Electrophoretic profile (SDS-PAGE) of proteins in the venom (Lane 2), peak 1 (Lane 3) and peak 2 (Lane 4). Lane 1 — molecular mass markers. Silver-stained 15% polyacrylamide gel. (C) Anion exchange chromatography of the second caseinolytic peak from the first step. The sample was applied to a 5 ml Hitrap Sepharose Q column equilibrated with 0.01 M Tris–HCl, pH 8.0, containing 0.01 M CaCl2. The column was eluted (0.5 ml/min) with a linear gradient of NaCl (0–0.5 M) and the elution profile was monitored at 280 nm. Fractions of 0.5 ml were collected. (■) protein content, (●) caseinolytic activity. The black bar in the first peak corresponds to the elution of BJ-PI2. (D) SDS-PAGE of purified BJ-PI2. Samples were run in a 15% gel and then silver stained. Lane 1 — molecular mass markers, Lane 2 — purified BJ-PI2. (E) RP-HPLC of BJ-PI2. Proteins were eluted with a linear gradient (0–100%) of acetonitrile in 0.1% TFA and the elution profile was monitored at 280 nm. (F) Molecular mass of BJ-PI2 in non-reducing and reducing conditions. Lane 1 — non-reducing conditions, Lane 2 — in the presence of 0.1% (w/v) dithiothreitol (DTT) and Lane 3 — in the presence of 0.1% (w/v) β-mercaptoethanol. Reducing agents did not alter the molecular mass of BJ-IP2, indicating that the protein was a monomer. M — molecular mass markers (in kDa): 97 — phosphorylase b, 66 — albumin, 45 — ovalbumin, 30 — carbonic anhydrase, 20.1 — soybean trypsin inhibitor, 14.4 — α-lactalbumin. Coomassie blue stained gel.

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100

%

22953.00

23194.00

22840.50 22712.00

mass

0 14000 16000 18000 20000 22000 24000 26000 28000 30000 32000 34000 36000 38000 40000 42000

Fig. 2. Molecular mass determination of native (non-reduced) BJ-PI2 by electrospray-ionization (ESI) mass spectrometry. The spectrum was obtained with a Quadrupole time-of-flight mass spectrometer (Q-TOF Ultima, Waters). Deconvolution of the spectrum yielded an isotope-averaged molecular mass of 23080.5 Da for the most intense signal.

according to the manufacturer's instructions. The first dimension was run at 6 °C using a three-phase electrophoresis program: 300 V for 1 min, 3500 V for 1.5 h and 3500 V for 3.5 h. Prior to running the second dimension, the IPG strips were placed in glass tubes and the proteins in the strip were reduced and alkylated by sequential incubation in the following solutions: (a) 0.05 M Tris–HCl, pH 8.8, 2% SDS, 30% glycerol, 6 M urea, 0.002% bromophenol blue (equilibration buffer — EB), (b) 10 mg of dithiothreitol/ml in EB and then (c) a solution of iodoacetamide (25 mg/ml) in EB. The strips were then applied directly to 15% SDS polyacrylamide gels (14 cm ×16 cm) for second-dimension electrophoresis at 300 V for 6 h. The gels were subsequently fixed and stained with silver. 2.6. In-gel protein digestion and mass spectrometric protein identification by LC-MS/MS The protein band was excised and in-gel trypsin digestion was done according to Hanna et al. [33]. An aliquot (4.5 μl) of the resulting peptide

A

mixture was injected into a trap column (180 μm i.d.×20 mm) packed with C18 chromatographic medium (Waters, Milford, MA, USA) for desalting with 100% solvent A (0.1% formic acid) at 5 μl/min for 3 min. Peptides were then eluted onto an analytical C18 column (75 μm i.d. × 100 mm) (Waters) using a 20 min gradient at a flow rate of 600 nl/min where solvent A was 0.1% formic acid and solvent B was 0.1% formic acid in acetonitrile. The gradient was 0–80% acetonitrile in 0.1% formic acid over 20 min. A QTOF Ultima mass spectrometer (Waters) was used to acquire spectra. Spray voltage was set at 3.1 kV and the instrument was operated in data dependent mode in which one full MS scan was acquired in the m/z range of 200–2000 followed by MS/MS acquisition using collision induced dissociation of the three most intense ions from the MS scan. A dynamic peak exclusion was applied to avoid the same m/z being selected for the next 120 s. The resulting fragment spectra were processed using ProteinLynx™ software (Waters, USA) and pkl* files were created and searched using the MASCOT search engine (Matrix Science, UK) against the SwissProt database restricted to ‘bony vertebrates’ sequences with a parent

B

Fig. 3. 2D electrophoresis of B. jararaca venom (300 μg) (A) and BJ-PI2 (B) (50 μg). Isolectric focusing gels strips (13 cm; pH 3–10 linear) were immersed in rehydration solution (250 μl) containing 7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer, 1% bromophenol blue and venom prior to use. The first dimension (isoelectric focusing) was done in three steps (300 V for 1 min, 3500 V for 1.5 h and 3500 V for 3.5 h). The second dimension was done in 15% SDS-PAGE gels (14 cm × 16 cm) run at 300 V for 6 h. The gels were silver stained. The bar in (A) corresponds to the position of BJ-PI2.

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Table 1 Summary of the purification of BJ-PI2. Purification step

Total protein (mg)

Total activity (units)a

Specific activity (units/mg)

Purification (fold)

Protein yield (%)

Venom Gel filtration (Superdex 75) Ion exchange (Sepharose Q)

103.8 ± 3.1 11.4 ± 0.4 0.469 ± 0.21

1302 ± 333 629 ± 124 93.9 ± 4.9

12.6 ± 3.6 54.6 ± 8.9 200.2 ± 10.6

1 4.3 ± 0.7 15.9 ± 0.8

100 11.8 ± 0.3 0.45 ± 0.18

a

Proteolytic activity with casein as substrate. The values are the mean ± SD of three purifications.

tolerance of 1.2 Da and fragment tolerance of 0.6 Da. Iodoacetamide derivatives of cysteine and oxidation of methionine were specified in MASCOT as fixed and variable modifications, respectively. 2.7. Protein quantification Protein concentrations were determined by the method of Lowry et al. [34] using bovine serum albumin as the standard. 2.8. Enzymatic assays 2.8.1. Caseinolytic activity Caseinolytic activity was assayed according to Kunitz [35]. Briefly, 1.9 ml of 1% casein in 0.1 M Tris–HCl, pH 7.8, was incubated with 0.1 ml of sample (50 μg of venom or purified protein) for 20 min at 37 °C. The reaction was stopped by adding 2 ml of 5% trichloroacetic acid and the tubes then stored on ice for 30 min prior to centrifugation (2500 g, 15 min, 25 °C). The absorbance of the supernatant was read at 280 nm in a Beckman DU800 spectrophotometer. One unit of activity was defined as an increase in absorbance (A280nm) of 0.001/min. 2.8.2. Esterase activity Esterase activity was assayed using Nα-p-tosyl-L-arginine ester (TAME) [36]. The reaction mixture consisted of 1.5 ml of substrate (1 mM TAME in 0.1 M Tris–HCl, pH 7.8), 1.4 ml of 0.1 M Tris–HCl, pH 7.8, and 0.1 ml of sample (40 μg venom or 40 μg purified protein). The mixture was incubated for 10 min at 25 °C after which the absorbance was measured at 253 nm. One unit of activity was defined as an absorbance increase of 0.001/min. 2.8.3. Elastase activity Elastase activity was assayed using elastin-Congo red and n-tboc-L-alanine p-nitrophenol ester as substrates. For the elastin-Congo red assay [37], the substrate was suspended (1 mg/ml) in 0.05 M sodium phosphate, pH 7.2, and 100 μl of this suspension was incubated with 100 μl of sample containing 10 μg of venom or 40 μg of purified protein for 18 h at 37 °C. After centrifugation (10,000 g, 15 min, 8 °C), the absorbance of the supernatant was read at 490 nm. One unit of activity was defined as an increase in absorbance (A490nm) of 0.001/min.

Activity towards the synthetic substrate n-t-boc-L-alanine pnitrophenyl ester was assayed according to Berlov et al. [38]. The assay mixture consisted of 150 μl of 0.05 M phosphate buffer, pH 7.2, containing substrate (0.2 mM) and 50 μl of buffer (blank) or sample (10 μg of venom or 40 μg of purified protein diluted in buffer) in a 96-well plate that was incubated at 37 °C for 30 min during which the increase in absorbance at 348 nm was measured in a SpectraMax340 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The amount of product formed was determined from a standard curve of p-nitrophenol and one unit of activity corresponded to the release of 1 mmol of p-nitrophenol/min. 2.8.4. Fibrinogenolytic activity Fibrinogenolytic activity was assayed by incubating 100 μl of plasminogen-free bovine fibrinogen (5 mg/ml; dissolved in 0.1 M Tris–HCl, pH 7.5, containing 0.1 M NaCl) with 100 μl of sample (10 μg of venom or purified protein) at 37 °C. At predetermined times (0, 5, 10 and 30 min, and 1, 2, 4 and 6 h) after sample addition, 20 μl aliquots were withdrawn from the incubation mixture and transferred to polypropylene Eppendorf tubes containing 20 μl of 2% β-mercaptoethanol and 2% SDS. After mixing, the samples were loaded onto 12.5% polyacrylamide gels and the proteins separated by SDS-PAGE (see Section 2.4); the gels were subsequently stained with Coomassie brilliant blue R250 [39]. Undegraded fibrinogen and molecular mass markers were included in the runs to allow assessment of fibrinogen chain digestion. In some assays, the purified protein was treated with protease inhibitors (10 mM EDTA, 5 mM 1–10 phenanthroline or 5 mM PMSF) for 10 min at 37 °C prior to incubation with fibrinogen. The remainder of the assay was then done as described above. 2.8.5. Fibrinolytic activity Fibrinolytic activity was assayed as described by Maruyama et al. [19]. Petri dishes were poured with molten 1% agarose in 0.1 M Tris–HCl, pH 7.5, containing 0.1 M NaCl and 0.5% plasminogen-free bovine fibrinogen. Immediately prior to pouring, 30 μl of thrombin (1 U/μl) was added to the mixture to clot the fibrinogen. Subsequently, 10 μl of venom or purified protein was added to wells cut in the agarose followed by incubation for 24 h at 37 °C. The diameters of the resulting halos were measured. One unit of activity corresponded to a halo diameter of 1 cm. 2.9. Biological activities

Table 2 Mass spectrometric data for the tryptic peptides of BJ-PI2. m/z observed (precursor ion) z

Peptide sequencea

Ion scoreb Deltac

422.74 449.75 547.73 782.36 847.40 691.33 548.75 448.89

DXXBVEK ERDXXPR YNSNXNTXR VHEMVNTXNGFFR YXEXAVVADHGMFTK TXTSFGEWRER TXTSFGEWR BSVAVVMDHSXK

44 50 63 62 85 53 57 18

a b c

+2 +2 +2 +2 +2 +2 +2 +3

X indicates Leu/Ile, B indicates Gln/Lys and M indicates oxidized Met. Mascot ion score. Difference between theoretical and observed mass.

−0.0185 −0.0129 −0.0193 −0.0601 −0.0531 −0.0330 −0.0315 −0.0242

2.9.1. Hemorrhagic activity Hemorrhagic activity was assayed according to Theakston and Reid [40]. Male Wistar rats were anesthetized with sodium thiopental (30 mg/kg, i.p.) and the dorsal region was shaved. Venom (4.5, 13.5 and 45.0 μg) or purified protein (10 and 40 μg) diluted in 0.01 M Tris–HCl, pH 8.0, containing 0.01 M CaCl2 was injected intradermally (fixed volume of 100 μl) and control sites were injected with Tris–HCl/CaCl2 buffer alone. After 24 h, the rats were killed with an overdose of isoflurane, the dorsal skin was removed and the diameter of the hemorrhagic halo on the inner surface of the skin was measured. The minimum hemorrhagic dose (MHD) was defined as the amount of venom or enzyme capable of producing a hemorrhagic halo 1 cm in diameter.

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2.9.2. Myotoxicity Male Swiss mice were injected with B. jararaca venom (40 μg), purified protein (40 μg) or 0.01 M Tris–HCl, pH 8.0, containing 0.01 M CaCl2 (control) in the right gastrocnemius muscle. At 3, 6, 12 and 24 h post-injection the mice were killed with isoflurane and blood samples were obtained for the quantification of plasma creatine kinase (CK) activity with commercial kits (LaborLab, Guarulhos, SP, Brazil); the gastrocnemius muscle was also removed at these same intervals for histological analysis.

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clot formation was monitored for 30 min at 650 nm in a SpectraMax 340 microplate reader (Molecular Devices). 2.10. Statistical analysis The results were expressed as the mean ± SD. Statistical comparisons were done using Student's t-test or ANOVA followed by the Bonferroni test. Values of p b 0.05 indicated significance. 3. Results

2.9.3. Vascular permeability assay Male Wistar rats were anesthetized with sodium thiopental (50 mg/kg, i.p.) and maintenance doses were administered when required. Local plasma protein extravasation was measured in the shaved dorsal skin, in response to intradermally injected B. jararaca venom (3–10 μg/site), purified protein (1–30 μg/site) or Tyrode solution (100 μl) [41]. Agents were injected in a random order, according to a balanced site pattern. Plasma protein extravasation was measured by the accumulation of intravenously (i.v.) injected 125I-human serum albumin (2.5 μCi/rat) with Evan's blue dye (20 mg/kg) serving as a visual marker. After 30 min, a cardiac blood sample (1 ml) was obtained from anesthetized rats that were then killed with an anesthetic overdose. The blood was transferred to polypropylene tubes containing 100 μl of heparin and then centrifuged (10,000 g, 8 min, room temperature) to obtain plasma. The dorsal skin was removed from the rats and the injected sites were punched out and counted for radioactivity, together with plasma samples, in a gamma-counter. Plasma extravasation was expressed as the volume (μl) of plasma accumulated at each skin site compared to total counts in 1 ml of plasma. In some experiments, the purified protein was preincubated with 5 mM 1,10-phenanthroline for 10 min at 37 °C prior to injection into dorsal rat skin. 2.9.4. Coagulant activity 2.9.4.1. Fibrinogen clotting in vitro. The ability of BJ-PI2 to clot fibrinogen in vitro was assayed by incubating 180 μl of plasminogen-free bovine fibrinogen (5 mg/ml; dissolved in 0.1 M Tris–HCl, pH 7.5, containing 0.1 M NaCl) with 20 μl of sample (40 μg of venom, 40 μg of BJ-PI2 or 20 U of thrombin) and the time required to clot at 37 °C was recorded. 2.9.4.2. Clotting of rat citrated plasma. The coagulant activity of venom and BJ-PI2 was examined using rat citrated platelet-poor plasma (PPP). Rat arterial blood was collected into polypropylene tubes containing 3.8% sodium citrate (anticoagulant:blood ratio 1:9, v/v). Platelet-rich plasma (PRP) was obtained by centrifugation (400 g, 20 °C, 15 min); PRP was then centrifuged (800 g, 20 °C, 13 min) to obtain PPP that was used in the coagulation test. For the assay, 190 μl of PPP was incubated at 37 °C with 10 μl of sample (thrombin: 0.3 U; venom: 40 μg; PI-BJ2: 10 or 40 μg) and the time required for clot formation was recorded. 2.9.4.3. Plasma recalcification. The influence of BJ-PI2 on the plasma recalcification time was assayed as described by Berger et al. [31], with some alterations. Briefly, platelet poor plasma (PPP) was obtained from citrated rat blood by centrifugation (3000 g, 15 min, room temperature) and 50 μl aliquots were transferred to a 96-well microplate and incubated with 50 mM Tris–HCl, pH 7.4 (control) or BJ-PI2 (5 or 10 μg) in Tris–HCl buffer at 37 °C. After a 5 min incubation with BJ-PI2, 10 μl of 250 mM CaCl2 was added to each well and

3.1. Purification of BJ-PI2 from B. jararaca venom Fractionation of B. jararaca venom on Superdex 75 resulted in four peaks, two of which had caseinolytic activity (Fig. 1A). The second of these peaks contained proteins with molecular masses of 20–35 kDa (by SDS-PAGE) (Fig. 1B) and was used in subsequent purifications. Fractionation of the second peak by anion exchange chromatography (Hitrap Sepharose Q) yielded five peaks, the first of which eluted before the salt gradient and contained caseinolytic activity (Fig. 1C); reverse-phase HPLC of this peak confirmed its purity (Fig. 1E). SDS-PAGE of this peak revealed a single protein band of 25.2 kDa (Fig. 1D) that was unaltered by reducing conditions (DTT or β-mercaptoethanol) (Fig. 1F). Mass spectrometry yielded a series of closely grouped signals that differed by a maximum of 482 Da; the most intense of these signals corresponded to a mass of 23.08 kDa (Fig. 2), which was similar to the value of ~ 25 kDa obtained by SDS-PAGE. Two-dimensional gel electrophoresis indicated that the protein was basic (pI ~ 8) (Fig. 3). Table 1 summarizes the purification protocol. BJ-PI2 corresponded to 0.45% of the venom protein and had a specific activity of 200.2 ± 10.6 units/mg (venom specific activity: 12.6 ± 3.6 units/mg; purification factor of ~ 16%, based on the proteolytic/caseinolytic activity). 3.2. Identification of BJ-PI2 by mass spectrometry of tryptic fragments Mass spectrometric analysis of a tryptic digest of the purified protein yielded eight MS/MS spectra with low background noise that provided information on the b and y-ion series. A database searches identified the fragments YIELAVVADHGMFTK, YNSNLNTIR, VHEMVNTLNGFFR, DLIKVEK, TLTSFGEWR, TLTSFGEWRER, ERDLLPR and QSVAVVMDHSKK. Table 2 shows the m/z and z of the precursor ions and other information for the peptide fragments identified by mass spectrometry. These fragments showed highest homology with the SVMP insularinase A from Bothrops insularis (UniProtKB/Swiss-Prot accession number Q5XUW8.1; [42]) but also shared homology with the B. jararaca SVMP bothrojaractivase (accession number P0C7A9.1; [31]); less similarity was observed with the B. jararaca SVMPs BJ-PI (peptide sequences from [24]) and jararafibrases II and IV (accession numbers Q98SP2.1 and P0C6S6.1, respectively; [19,43]); there was also some homology with BaP1, a P-I SVMP from B. asper venom (accession number P83512.2; [44]) (Fig. 4). Based on this analysis and on additional findings described below, this protein was identified as a P-I SVMP and named BJ-PI2, to avoid confusion with the previously identified BJ-PI, another P-I class metalloproteinase in B. jararaca venom [24]. 3.3. Enzymatic and biological activities In addition to the proteolytic activity indicated in Section 3.1, BJ-PI2 (40 μg) degraded the substrate elastin-Congo red (128.8±5.2 units/mg

Fig. 4. Sequences of BJ-PI2 fragments identified by mass spectrometry and a database search: comparison with other Bothrops P-I SVMPs. In gray, fragments showing homology with insularinase A and bothrojaractivase identified in this work. Amino acids with a single underline — tryptic fragments identified by Zelanis et al. [45], amino acids in bold — tryptic fragments identified by Zelanis et al. [46] and amino acids in bold with a double underline — amino acids in bothrojaractivase that differ from BJ-PI2. The UniProtKB/Swiss-Prot accession numbers for the proteins in this table are P83512.2 for BaP1 [44], Q5XUW8.1 for insularinase A [42], P0C7A9.1 for bothrojaractivase [31], Q98SP2.1 for jararafibrase II [19,43] and P0C6S6.1 for jararafibrase IV [43]. Note that the jararafibrase II sequence is contained within the metalloproteinase domain of the gene encoding for the disintegrin bothrostatin [47]. The fragment sequences for BJ-PI are from Oliveira et al. [24] as they are not available through the UniProtKB/Swiss-Prot database. X indicates Leu/lle, B indicates Gln/Lys.

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compared to 413.7 ± 34.0 units/mg for 40 μg of venom; n = 3) but was less active towards the synthetic elastase substrate n-t-boc-L alanine p-nitrophenyl ester (10.8± 7.7 units/mg compared to 131.5 ± 27.7 units/mg for 40 μg of venom; n = 3). BJ-PI2 had no esterase activity (venom esterase activity: 282.0 ± 22.6 units/mg; n = 3). BJ-PI2 (40 μg) did not clot fibrinogen in vitro, in contrast to B. jararaca venom (40 μg) and thrombin (20 U) which produced clots in 12±1 s and 20±1 s, respectively (n=3 each). In agreement with this finding, BJ-PI2 (40 μg) also did not clot rat citrated plasma, in contrast to B. jararaca venom (40 μg; 42±11 s, n =5) and thrombin (0.3 U; 281±38 s, n=5). Although BJ-PI2 (40 μg) did not clot rat citrated plasma it was nevertheless fibrinogenolytic and cleaved the α chain of fibrinogen very rapidly (within 10 min) while the β chain was degraded more slowly (within 30 min); there was limited digestion of the γ chain (Fig. 5A). This fibrinogenolytic activity was inhibited by 1,10phenanthroline and EDTA but not by PMSF (Fig. 5B), indicating that BJ-PI2 is a metalloproteinase (rather than a serine proteinase). BJ-PI2 (10 μg) attenuated the rate of coagulation during plasma recalcification, as judged from the inclination of the ascending portion of the curve, and markedly reduced the extent of coagulation without affecting the time for the initiation of coagulation; a lower amount of BJ-PI2 (5 μg) did not affect coagulation in response to recalcification (Fig. 6). BJ-PI2 (10 μg) was also fibrinolytic (halo of 3.2±0.7 cm in the fibrin plate assay compared to 6.3±0.0 cm for 10 μg of venom). BJ-PI2 caused a dose-dependent increase in vascular permeability in rat dorsal skin (Fig. 7A) that was abolished by preincubating the protein with 1,10-phenanthroline (Fig. 7B). BJ-PI2 (40 μg) was not hemorrhagic in rat dorsal skin (halob 0.1 cm) whereas a venom dose of 40 μg produced a hemorrhagic halo of 1.2 ±0.2 cm (the MHD for venom was 20.7 μg). The intramuscular injection of BJ-PI2 (40 μg) did not cause myonecrosis, assessed either as an increase in circulating CK levels or as histological damage; in contrast, venom (40 μg) produced a marked increase in CK levels and extensive histological damage (hemorrhage and necrosis) (Fig. 8). Although BJ-IP2 was not hemorrhagic, it caused marked infiltration of inflammatory cells 3, 6 and 12 h after injection compared to control (saline) and venom-treated mice. 4. Discussion B. jararaca is responsible for most bites by Bothrops spp. in Brazil, particularly in the southeast of the country. Envenoming by this species results in local edema, hemorrhage and necrosis, while the most common systemic manifestations are hemostatic disturbances (coagulopathy and platelet dysfunction) and systemic hemorrhage that are shared with other species of the genus [6–10]. Several of these local and systemic effects are mediated by SVMPs [2,4,7,10,11]. The current classification of SVMP includes three major classes (P-I, P-II and P-III) and some subclasses, with the most hemorrhagic toxins belonging to P-III class and those with weakest activity belonging to P-I class [1,2,4,48]. Various P-I class SVMPs have been purified and characterized from B. jararaca venom, including jararafibrases II, III and IV [19,30], bothrojaractivase [31] and BJ-PI [24]. These SVMPs and related proteins isolated from other Bothrops spp. have a molecular mass of ~ 20–27 kDa and are generally fibrino(geno)lytic, but show variable hemorrhagic and myotoxic activities (Table 3). The results described here and a comparison with data in the literature indicate that we have identified a new non-hemorrhagic P-I metalloproteinase (BJ-PI2, ~ 25 kDa by SDS-PAGE and 23 kDa by mass spectrometry) from B. jararaca venom. The protein was purified in two chromatographic steps (gel filtration on Superdex 75 followed by anion exchange chromatography on Sepharose Q) and yielded a single band in SDS-PAGE and a single symmetrical peak in HPLC. Mass spectrometry revealed a series of associated peaks (maximum mass difference of 482 Da), possibly indicative of closely related isoforms that differ in a few amino acids or in post-translational modifications. BJ-PI2 was inhibited by EDTA and 1,10-phenanthroline

(metalloproteinase inhibitors) but not by PMSF (a serine proteinase inhibitor); this inhibition profile was similar to that for other Bothrops P-I SVMPs [52–55,57,58,63]. The analysis of tryptic fragments indicated that BJ-PI2 shared greatest homology with insularinase A, a P-I class SVMP isolated from B. insularis venom [42]. Previous transcriptomic [65,66] and proteomic [45,46] analyses of B. jararaca venoms from neonates and adults have also identified proteins related to insularinase A, but this protein has not previously been isolated and studied. Analysis of Table 2 and Fig. 4 shows that seven of the eight fragments identified here were previously identified by Zelanis et al. [45,46]; the eighth (BSVAVVMDHSBB) fragment identified here was new. Fragments not detected here but identified in previous work [45,46] include ASXANXEVWSB, SCXMASTXSB, ABCAEGXCCDBCR, CTGBSADCPR and GDNPDDRCTGBSADCPR (see Fig. 4). Together, these sequences indicate that BJ-PI2 is structurally related to insularinase A. BJ-PI2 accounted for 0.45% of the total venom protein. This yield was similar to that for other P-I SVMPs from B. jararaca venom (except for BJ-PI), but lower than for several other Bothrops P-I SVMPs (Table 3). Adult specimens of B. jararaca can yield up to 150 mg of venom [67–69] that would theoretically contain ~0.675 mg or 675 μg of BJ-PI2. Based on the results described above, this amount of BJ-PI2 would be sufficient to produce the local effects observed in this work since in most of the experiments only 10–40 μg of the protein was used. Envenoming by B. jararaca is characterized by hemostatic disturbances, including blood incoagulability and platelet dysfunction [7,70,71], and proteins capable of interfering with the coagulation cascade and platelet aggregation have been identified in the venom of this species [18,72]. Although BJ-PI2 did not clot fibrinogen or rat citrated plasma, i.e., no thrombin-like activity leading to clot formation, this protein was strongly fibrinogenolytic in vitro, with the preference for chain cleavage being α >β. This chain selectivity was similar to that observed for bothrojaractivase [31] and jararafibrase II [19] from B. jararaca and Batx-I from Colombian Bothrops atrox [53]. Indeed, most Bothrops P-I SVMPs are α/β fibrinogenases, although some of them are essentially α-fibrinogenases (Table 3). This divergence in the susceptibility of fibrinogen chains to degradation may partly reflect variations in the SVMP concentration and incubation times tested. The strong fibrinogenolytic activity of BJ-PI2 resulted in a decrease in the rate and extent of recalcification that reflected substrate depletion by this protein. Like other Bothrops P-I SVMPs (Table 3), BJ-PI2 was also fibrinolytic. Together, these activities could contribute to coagulopathy after envenoming by B. jararaca. Although some P-I SVMPs are inhibited by α2-macroglobulin [52,73] this was probably not the case with BJ-PI2 since in plasma the protein was still able to degrade fibrinogen and markedly reduce the extent of coagulation during plasma recalcification. P-I class SVMPs vary in their ability to cause hemorrhage (Table 1), and those that do cause hemorrhage are only weakly so, especially when compared to P-III SVMPs [4]. As shown here, in contrast to the venom, BJ-PI2 was not hemorrhagic and therefore probably contributes little to this effect of the venom. However, hemorrhagic activity may vary with the route of administration. Thus, for example, neuwiedase (a P-I SVMP from Bothrops neuwiedi) is not hemorrhagic in mouse skin or gastrocnemius muscle but causes hemorrhage when applied topically to exposed mouse cremaster muscle (as assessed by intravital microscopy) and in lungs (but not other internal organs) after i.v. administration [64]. The limited hemorrhagic activity of P-I SVMPs compared to P-III SVMPs has been attributed to the poor ability of the former toxins to bind and cleave basement membrane proteins such as collagen IV [4,74] and their susceptibility to inhibition by circulating proteins such as α2-macroglobulin [4]. The presence of carbohydrate moieties and of disintegrin-like, cysteine-rich and C-type lectin domains in P-III SVMPs may also be important factors in their hemorrhagic activity [4,25]. The ability to cause hemorrhage is frequently related to the capacity to degrade specific vessel wall components and SVMPs that

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Control (Tris-HCl)

A kDa

M

1817

BJPI-2 (5 μg)

BJPI-2 (10 μg)

125

97

66 45

Extent of clotting (%)

α β γ

30

20.1

100

75

50

25

0 0

14.4 0

10

30

60

120

240

kDa

M

1

2

3

4

5

97 66 45

400

600

800 1000 1200 1400 1600 1800 2000

Time (s)

360

Incubation time (min)

B

200

Fig. 6. BJ-PI2 (10 μg) attenuates the rate and extent of coagulation during plasma recalcification. Plasma samples (50 μl) were preincubated with 50 mM Tris–HCl, pH 7.4 (control) or BJ-PI2 (5 μg or 10 μg in Tris–HCl buffer) in a 96-well microplate at 37 °C for 5 min prior to the addition of 10 μl of 250 mM CaCl2 to each well. Clot formation was monitored for 30 min at 650 nm in a microplate reader. The results are expressed as a percentage of the maximum clotting observed after recalcification in control plasma samples treated with Tris–HCl buffer alone. The points are the mean ± SD (n = 3 independent experiments using plasma from three rats).

α β γ

30

20.1

14.4 Fig. 5. Fibrinogenolytic activity of BJ-PI2. (A) Time-course of fibrinogenolytic activity of BJ-PI2 (10 μg). At the times indicated on the X-axis fibrinogen chain degradation was assessed by withdrawing samples from the fibrinogen-BJ-PI2 mixture (incubated at 37 °C) and analyzing them by SDS-PAGE. BJ-PI2 cleaved the Aα chain first, followed by the Bβ chain; the γ chain was relatively resistant to digestion. (B) Influence of protease inhibitors on fibrinogenolytic activity of BJ-PI2 (10 μg). Lane 1 — fibrinogen incubated without BJ-PI2 showing all of the chains (α, β and γ), Lane 2 — fibrinogen+BJ-PI2, Lane 3 — fibrinogen+BJ-PI2 pre-incubated with 5 mM 1,10-phenanthroline, Lane 4 — fibrinogen+BJ-PI2 pre-incubated with 5 mM PMSF and Lane 5 — fibrinogen+BJ-PI2 preincubated with 1 mM EDTA. The incubation time of fibrinogen with BJ-PI2 in these experiments was 360 min. In both panels, the samples were run in 12.5% polyacrylamide gels (SDS-PAGE) that were subsequently stained with Coomassie brilliant blue. M — molecular mass markers (in kDa). See Fig. 1 legend for marker identification.

are unable to disrupt vessel walls by degrading the ECM generally provoke little or no hemorrhage. On the other hand, a synergistic action between hemostatic disturbances and direct damage to the vessel by hemorrhagic toxins can result in hemorrhage [4,11]. In the case of BJ-PI2, although this protein had fibrino(geno)lytic activity (that could cause coagulation disturbances), it did not cause hemorrhage, possibly because of limited degradation of ECM proteins, as suggested for non-hemorrhagic P-I SVMPs [4,74]. Although BJ-PI2 was active towards the synthetic elastase substrate n-t-boc-L-alanine p-nitrophenyl ester and the natural substrate elastin-Congo red this activity probably reflected a non-specific proteolytic action of BJ-PI2 rather than true elastase activity since B. jararaca venom shows no activity towards the highly specific elastase substrates n-methoxysuccinyl-Ala-Ala-Pro-Valp-nitroanilide and n-succinyl-Ala-Ala-Ala-p-nitroanilide (unpubl. obs.). Bothrops venoms are frequently myotoxic and this activity is mediated primarily by myotoxic PLA2, with SVMPs exerting a less

Fig. 7. Increase in vascular permeability caused by BJ-PI2. (A) Dose‐response curve to BJ-PI2. (B) Inhibition of the increase in vascular permeability by 1,10-phenanthroline. BJ-PI2 (10 μg) was pre-incubated with 5 mM 1,10-phenanthroline for 10 min at 37 °C prior to injecting into mouse dorsal skin. Pre-incubation with 1,10-phenanthroline significantly (pb 0.05) attenuated the increase in vascular permeability. In (A) and (B), the points or columns are the mean±SD of n=5. *pb 0.05 compared to Tyrode alone and 1,10-phenanthroline (phen) alone. #pb 0.05 compared to BJ-PI2+phen.

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jararacussu [54] and BnP1 from B. neuwiedi [62], are also not myotoxic and contribute little to this action in their respective venoms. Although not hemorrhagic or myotoxic, BJ-PI2 markedly stimulated the infiltration of inflammatory cells 3, 6 and 12 h after injection. This finding agrees with Rodrigues et al. [64] who showed that

prominent role. As with hemorrhagic activity, P-I SVMPs vary in their ability to cause myonecrosis (Table 3). As shown here, BJ-PI2 was not myotoxic (as assessed by CK release and histological damage) and is therefore unlikely to be an important venom component in this response. Other Bothrops P-I SVMPs, such as BjussuMP-II from Bothrops

A

B

C

D

* *

* * H H E

* * *

*

F

*

H

G

*

*

* *

H

I

J

H

* *

H H Fig. 8. Assessment of tissue damage by BJ-PI2. (A) Circulating plasma CK levels in mice injected intramuscularly with B. jararaca venom (40 μg), BJ-PI2 (40 μg) or 0.01 M Tris–HCl, pH 7.5 (20 μl, fixed volume used to inject venom or toxin). Mice were inoculated in the gastrocnemius muscle and blood samples were obtained at 0, 3, 6, 12 and 24 h for the quantification of plasma CK levels. The points are the mean ± SD (n = 4). (B–J) Histological changes in gastrocnemius muscle injected with saline (control; B,E,H), venom (C,F,I) or BJ-PI2 (D,G,J). At the times indicated above, the muscle was removed, fixed in buffered 10% formol and processed for histological analysis. Sections 5 μm thick were stained with hematoxylin-eosin (HE). (B–D) represent mice 3 h post-injection, (E–G) 6 h post-injection and (H–I) 12 h post-injection. *, H and arrows represent myonecrotic fibers, hemorrhage and inflammatory cells, respectively.

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Table 3 Comparison of the properties of BJ-PI2 and other P-I metalloproteinases isolated from B. jararaca and other Bothrops spp. venoms. Species

Enzyme

Fibrinolytic

Fibrinogenolytic

Hemorrhagicb

Edematogenic

Myonecrotic

Yield (%)

Mass (kDa)

References

B. jararaca

BJ-PI2 BJ-PI Bothrojaractivase Jararafibrase II Jararafibrase III Jararafibrase IV BaP1 BH2 Atroxlysin-I Batx-1 Insularinase A BjussuMP-II Leucurolysin-a BmHF-1 BthMP BmooMPα-I Moojeni protease A BnP1 Neuwiedase

Yes

Yes Yes Yes Yes

No No

Yes

No

a

a

a

a

a

Yes Yes Yes Yes Yes Yes Yes No No No Yes Yes No No Yes Noc

a

a

0.45 14 0.19 0.35

a

a

a

a

a

Yes

Yes

a

a

a

a

a

a

a

Yes

5.1 45

23.1 22.0 22.8 21.4 20.4 21.2 24.0 26.0 23.0 23.3 22.6 24.0 23.0 27.2 23.5 23.1 20.4 24.0 22.4

This work [24,25] [31] [19] [30] [30] [49,50] [51] [52] [53] [42] [54] [55,56] [57] [58] [59] [60,61] [62] [63,64]

B. asper B. atrox B. B. B. B. B.

insularis jararacussu leucurus marajoensis moojeni

B. neuwiedi

a

Yes Yes Yes Yes a

a a

Yes (α/β chains)

a

a

Yes

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

a

Yes Yes Yes a

Yes Yes Yes Yes Yes

(α/β chains) (α chain) (α/β chains) (α/β chains)

(α chain) (α/β chains) (α/β chains) (α/β chains) (α/β chains) (α chain) (α/β chains) (α/β chains) (α/β chains) (α/β chains) (α/β chains)

a

a

a

a

Yes Yes Yes Yes

No

a

a

a

a

5.4 8–10 2.3 8.7 2.4

No Yes

a

a

a

No Yes

Yes

a

4.0

a

Not determined/not reported. The toxins with activity are only weakly hemorrhagic. c No hemorrhage in mouse dorsal skin or gastrocnemius muscle, but bleeding observed by intravital microscopy after local application to mouse cremaster muscle and hemorrhage in lungs after i.v. injection [64]. b

although neuwiedase was not hemorrhagic and only mildly myotoxic this P-I SVMP nevertheless caused a marked inflammatory reaction in mouse gastrocnemius muscle after i.m. administration. Similarly, Gomes et al. [58] have shown that BthMP from Bothrops moojeni caused marked leukocyte infiltration in this same animal model, with a gradual return to normal after 24 h. The local inflammatory response seen with neuwiedase apparently involved enhanced cytokine (IL-1β, IL-6 and KC — a murine functional homolog of human IL-8) production (sustained for up to 8 h in the case of IL-1β) by skeletal muscle [75]; variable increases were also observed for IL-6, KC and TNFα in murine peritoneal adherent cells (MPACs) and C2C12 cells (a skeletal muscle cell line), depending on the cell type. BaP1, a hemorrhagic P-I SVMP from B. asper venom, also enhances IL-1 and IL-6 production (but not TNF-α or IFN-γ levels) in mouse skeletal muscle [15]. A similar action by BJ-PI2 could account for the ability of this toxin to increase inflammatory cell migration into muscle and enhance vascular permeability. In conclusion, we have isolated and characterized a new metalloproteinase (BJ-PI2) in B. jararaca venom. The actions of this toxin on vascular permeability and inflammatory cell migration indicate that BJ-PI2 is involved in these local responses in vivo. In contrast, BJ-PI2 appears to have little role in venom-induced hemorrhage and necrosis. In view of current interest in the hemostatic actions of snake venom components, the fibrin(ogen)olytic activity of BJ-PI2, coupled with its lack of hemorrhagic and necrotic activity, makes this protein a potentially interesting molecule for studies of its possible therapeutic application in thrombotic disorders. Role of funding source None of the funding agencies indicated in the Acknowledgments was involved in the study design, data collection, analysis and interpretation, or in the preparation and writing of the article and the decision to submit the paper for publication. Acknowledgements The authors thank José Ilton dos Santos for technical assistance. I.R.F.S. and A.L.R. were supported by an MSc and a PhD scholarship, respectively, from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), L.B. was supported by a PhD scholarship

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